A method for diluting rabbit semen and cryopreserving rabbit semen

Abstract

This invention provides a rabbit frozen semen diluent and a method for cryopreserving rabbit semen, belonging to the field of artificial insemination technology. The rabbit frozen semen diluent comprises the following components: tris(hydroxymethyl)aminomethane, citric acid, glucose, sucrose, antibiotics, dimethyl sulfoxide, egg yolk, water, and a pH adjuster. The method for cryopreserving rabbit semen includes the following steps: adding the aforementioned rabbit frozen semen diluent to rabbit semen to obtain a mixture, dispensing, equilibrating, and cryopreserving. The rabbit frozen semen diluent and method for cryopreserving rabbit semen provided by this invention can be used for cryopreserving rabbit semen at low temperatures, effectively reducing the damage to semen caused by low temperatures during cryopreservation, improving sperm motility and conception rate in female rabbits upon thawing, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of artificial insemination technology, specifically relating to a rabbit frozen semen diluent and its preparation method, as well as a method for cryopreserving rabbit semen. Background Technology

[0002] In the fields of life sciences, human medicine, and health research, laboratory animals share many similarities with humans or other animals in their physiological and pathological processes, and can serve as mutual references. To ensure more scientific, accurate, and reproducible animal experiments, various methods can be used to stably demonstrate certain physiological or pathological activities in standardized laboratory animals for experimental research. These standardized laboratory animals are called model animals. Rabbits, as a commonly used model animal, play a vital role in biomedical research, including reproductive physiology and embryology, immunology, cardiovascular and pulmonary heart disease, infectious diseases, and the establishment of transgenic disease animal models.

[0003] Artificial insemination is a crucial method for achieving large-scale rabbit farming, enabling simultaneous breeding, farrowing, and market entry. This technique first requires collecting semen from male rabbits, examining its quality, diluting it with a diluent, and finally introducing the semen into the reproductive tract of estrus female rabbits, replacing natural mating. Artificial insemination increases semen utilization efficiency, reduces the number of male rabbits needed, decreases disease transmission, and improves conception rates in female rabbits.

[0004] Cryopreservation of semen plays a crucial role in extending the usability of specific sperm. However, sperm are highly sensitive to low temperatures, and the formation of intracellular ice crystals during cryopreservation is a major factor affecting sperm quality after thawing, leading to decreased sperm motility. Therefore, the selection of a cryopreservation diluent is extremely important. Currently, there is considerable research on cryopreservation diluents for pig, horse, cattle, and sheep semen, and these products are fully commercialized. However, research on cryopreservation diluents for rabbit semen is limited. There are currently almost no commercially available rabbit semen cryopreservation diluents on the market, and those that exist vary greatly in quality, resulting in low sperm motility after thawing, making it difficult to effectively conduct artificial insemination and reproductive practices.

[0005] Patent application CN114009427A discloses a rabbit semen cryopreservation diluent and its preparation method. The diluent uses water as a solvent and contains the following components added per 100 mL of water: 3-4 g of tris(hydroxymethyl)aminomethane, 1-2 g of citric acid, 0.5-1 g of glucose, 2-3 g of trehalose, 0.5-1 g of L-glutamic acid, 10-20 mL of dimethyl sulfoxide, and 2-4 mL of glycerol. The pH of the diluent is 6.5-7.5. This diluent can improve sperm motility to some extent after freezing; however, using this diluent for cryopreservation of rabbit semen still causes damage, and sperm motility upon thawing needs further improvement. Summary of the Invention

[0006] To address the aforementioned problems, the first objective of this invention is to provide a rabbit frozen semen diluent for cryopreservation, which can effectively reduce the damage to the semen caused by low temperature during cryopreservation and improve sperm motility and conception rate in female rabbits during thawing.

[0007] The second objective of this invention is to provide a method for cryopreserving rabbit semen, with the aim of promoting the application of rabbit cryopreservation semen technology in production and scientific research.

[0008] This invention provides a rabbit frozen semen diluent comprising the following components: tris(hydroxymethyl)aminomethane, citric acid, glucose, sucrose, antibiotics, dimethyl sulfoxide, egg yolk, water, and a pH adjuster;

[0009] The concentrations of tris(hydroxymethyl)aminomethane were 1.800–2.000 g / 50 mL, citric acid was 0.950–1.250 g / 50 mL, glucose was 0.325–0.725 g / 50 mL, sucrose was 0.25–0.65 g / 50 mL, antibiotics were 0.002–0.006 g / 50 mL, dimethyl sulfoxide was 1–8 ml / 50 mL, and egg yolk was 5–15 ml / 50 mL.

[0010] Furthermore, the antibiotic is kanamycin.

[0011] Further, the concentration of tris(hydroxymethyl)aminomethane is 1.850–1.950 g / 50 mL, the concentration of citric acid is 0.950–1.00 g / 50 mL, the concentration of glucose is 0.4–0.6 g / 50 mL, the concentration of sucrose is 0.35–0.55 g / 50 mL, the concentration of kanamycin is 0.003–0.005 g / 50 mL, the concentration of dimethyl sulfoxide is 3–5 mL / 50 mL, and the concentration of egg yolk is 7–12 mL / 50 mL.

[0012] Further, the concentrations of the tris(hydroxymethyl)aminomethane are 1.900 g / 50 mL, the concentrations of citric acid are 0.990 g / 50 mL, the concentrations of glucose are 0.525 g / 50 mL, the concentrations of sucrose are 0.428 g / 50 mL, the concentrations of kanamycin are 0.004 g / 50 mL, the concentrations of dimethyl sulfoxide are 5 mL / 50 mL, and the concentrations of egg yolk are 10 mL / 50 mL.

[0013] Furthermore, the pH value of the rabbit frozen semen diluent is 6.0–8.0.

[0014] Furthermore, the pH value of the rabbit frozen semen diluent is 7.0.

[0015] The present invention also provides a method for preparing the above-mentioned rabbit frozen semen diluent, the method comprising the following steps: mixing tris(hydroxymethyl)aminomethane, citric acid, glucose, sucrose, antibiotics, dimethyl sulfoxide and egg yolk, adding a pH adjuster to adjust the pH value, adding water to make up to a final volume, thereby obtaining the rabbit frozen semen diluent.

[0016] The present invention also provides a method for cryopreserving rabbit semen, which includes the following steps: adding the above-mentioned rabbit frozen semen diluent to rabbit semen to obtain a mixture, dispensing, equilibrating, and cryopreserving.

[0017] Furthermore, the volume ratio of rabbit semen to rabbit frozen semen diluent is 1:(1-4), the equilibration temperature is 2-6℃, and the time is 0.5-3h; the cryopreservation procedure is as follows: place the equilibrated aliquoted mixture at -85 to -105℃, then cool it down to -140 to -160℃ at a rate of -1 to -5℃ / min, and store it in a liquid nitrogen tank.

[0018] Furthermore, the equilibration temperature is 4°C and the time is 2 hours; the cryopreservation procedure is as follows: place the equilibrated aliquoted mixture at -95°C, then cool it down to -150°C at a rate of -3°C / min, and store it in a liquid nitrogen tank.

[0019] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0020] Compared with the known frozen semen diluent for rabbits in patent application CN114009427A, the frozen semen diluent for rabbits provided by this invention can effectively reduce the damage to semen caused by low temperature during cryopreservation and improve sperm motility and conception rate of female rabbits during thawing.

[0021] Compared with the cryopreservation method used in patent application CN114009427A, the method for cryopreserving rabbit semen provided by this invention, combined with the rabbit frozen semen diluent of this invention, is more conducive to reducing the damage to semen caused by low temperature during cryopreservation and improving sperm motility during thawing.

[0022] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0023] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0024] Unless otherwise specified, the raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0025] Example 1: Preparation of rabbit frozen semen diluent

[0026] Table 1. Formula for rabbit semen diluent

[0027] reagents dose Tris(hydroxymethyl)aminomethane (Tris) 1.900g Citric acid 0.990g glucose 0.525g sucrose 0.428g Kanamycin 0.004g Dimethyl sulfoxide (DMSO) 5ml yolk 10ml

[0028] After preparing the formula according to Table 1, add 1M sodium hydroxide solution to adjust the pH to 7.0, and add sterile water to make up to 50mL to obtain 50mL of rabbit frozen semen dilution.

[0029] Example 2: Method for cryopreserving rabbit semen

[0030] Semen was collected from male rabbits and diluted with rabbit frozen semen diluent (prepared in Example 1) at 4 times its volume. The semen was then dispensed into 0.25 ml straws, sealed with hemostatic forceps, and labeled. The straws were then placed in a refrigerator at 4°C for 2 hours to equilibrate. Next, the straws were cryopreserved using a CRYSAKYS PTC-9500 programmed freezing instrument. The program was set as follows: the instrument first cooled to -95°C, the straws were placed in the instrument, and then the temperature was lowered from -95°C to -150°C at a rate of -3°C / min. Once the target temperature was reached, the straws were cryopreserved in a liquid nitrogen tank.

[0031] The following experimental examples demonstrate the beneficial effects of the present invention.

[0032] Experimental Example 1: The effect of the rabbit frozen semen diluent of the present invention on sperm motility and pregnancy rate

[0033] 1. Experimental Methods

[0034] (1) Microscopic examination of viability

[0035] Semen of a 1.5-year-old male New Zealand white rabbit (1.3 ml) was collected and diluted with four times its volume of rabbit frozen semen diluent (prepared in Example 1). The semen was then dispensed into 0.25 ml straws, sealed with hemostatic forceps, and labeled. The straws were then placed in a freezer at 4°C for 2 hours to equilibrate. Next, a CRYSAKYS PTC-9500 programmed freezing system was used for cryopreservation. The program was set as follows: the instrument first cooled to -95°C, the straws were placed in the freezer, and then the temperature was lowered from -95°C to -150°C at a rate of -3°C / min. Once the target temperature was reached, the straws were cryopreserved in liquid nitrogen. After cryopreservation, one straw was thawed in a 37°C water bath for 15 seconds, and its motility was examined under a microscope.

[0036] The method for calculating sperm motility is as follows: After thawing a straw in a 37°C water bath for 15 seconds, take 10 μL of semen onto a glass slide, cover it with a coverslip, and evaluate the percentage of sperm that move linearly forward under a 400× inverted microscope.

[0037] (2) Insemination and birthing

[0038] Ten 1-year-old female rabbits were selected and artificially inseminated using rabbit semen thawed in step (1). The specific insemination details and the number of offspring were recorded.

[0039] 2. Experimental Results

[0040] (1) Microscopic examination of viability

[0041] Microscopic examination revealed that the semen motility was 60% after thawing.

[0042] (2) Insemination and birthing

[0043] Pregnancy and birthing: 6 animals became pregnant (38 offspring were born in total), with a pregnancy rate of 60%.

[0044] The success rate of natural mating during the same period was 75%, which proves that the semen frozen using the frozen semen diluent of this invention has high semen motility after thawing, and the results of artificial insemination are good. Its pregnancy rate can approach or reach the success rate of natural mating.

[0045] Experimental Example 2: Comparison of the effects of the rabbit frozen semen diluent of the present invention and existing rabbit frozen semen diluents on semen motility.

[0046] 1. Source of rabbit frozen semen diluent

[0047] Diluent 1: The rabbit frozen semen diluent prepared in Example 1 of this invention;

[0048] Diluent 2: Rabbit frozen semen diluent (pH = 7.3) prepared according to the formulation of Example 1 in patent application CN114009427A, as shown in Table 2:

[0049] Table 2. Formulation of patent application CN114009427A (per 50 mL sterile water)

[0050] reagents dose Tris(hydroxymethyl)aminomethane (Tris) 1.515g Citric acid 0.845g glucose 0.425g Trehalose 1.025g L-glutamic acid 0.370g Dimethyl sulfoxide 8ml glycerin 1.5ml

[0051] 2. Experimental Methods

[0052] 2.1 Fresh semen was collected from three male rabbits and mixed. The motility of the mixed semen was 90%. After collection, the gel in the semen was removed, and the mixture was frozen at 4°C for 90 minutes. At the same time, the diluent was stored at 4°C. 1.2.2 The refrigerated semen was mixed isothermally with the frozen semen diluent for the rabbits to be tested, at a mixing ratio of 1:1 or 1:4.

[0053] 2.3 After mixing, place the mixture at 4°C for 45 min to equilibrate, and then dispense it into 0.25 ml wheat tubes (pre-equilibrated at 4°C).

[0054] 2.4 Cryopreservation: There are two cryopreservation methods: (1) the cryopreservation program of the programmed cooling device in Example 2 of this invention; (2) the cryopreservation method described in patent application CN114009427A, which involves placing the equilibrated mixture 5cm above the liquid nitrogen surface for fumigation for 10 minutes, and then freezing it in liquid nitrogen after fumigation.

[0055] 2.5 Thawing: 50℃ water bath for 10 seconds.

[0056] 3. Experimental Results

[0057] 3.1 After mixing the semen and diluent isothermally in step 2.2, observe the motility under a microscope:

[0058] Diluent 1: When the semen and diluent are mixed at a 1:1 ratio, the sperm under microscopic examination all move in a straight line with almost no space between them, and the number of motile sperm accounts for 90% of the total. When the diluent is mixed at a 1:4 ratio, the sperm under microscopic examination all move in a straight line with spaces of varying sizes between them, each sperm is clearly distinguishable, and the number of motile sperm accounts for 90% of the total.

[0059] Diluent 2: When semen and diluent are mixed at a 1:1 ratio, microscopic examination reveals partial swaying movement of the sperm, with almost no space between the sperm, and the number of motile sperm accounts for 60% of the total; when mixed at a 1:4 ratio, all semen is dead.

[0060] 3.2 After equilibration at 4℃ for 45 minutes as per step 2.3, observe the viability under a microscope before dispensing.

[0061] Diluent 1: When the semen and diluent are mixed at a 1:1 ratio, the sperm under microscopic examination all move in a straight line with almost no space between them, and the number of motile sperm accounts for 80% of the total. When the diluent is mixed at a 1:4 ratio, the sperm under microscopic examination all move in a straight line with spaces of varying sizes between them, and each sperm is clearly distinguishable, and the number of motile sperm accounts for 60% of the total.

[0062] Diluent 2: When the semen and diluent are mixed at a 1:1 ratio, microscopic examination reveals that only a few sperm move in a straight line, while the rest sway or remain still. There is almost no space between the sperm, and the number of motile sperm accounts for 10% of the total. When mixed at a 1:4 ratio, all the semen dies.

[0063] 3.3 After freezing and thawing according to the cryopreservation procedure of the cooling device in Embodiment 2 of the present invention, the viability was observed under a microscope:

[0064] Diluent 1: When semen and diluent are mixed at a 1:1 ratio, microscopic examination of the semen shows that some sperm move in a straight line while the rest sway or remain still, with almost no space between the sperm and the number of motile sperm accounts for 50% of the total. When mixed at a 1:4 ratio, microscopic examination of the semen shows that some sperm move in a straight line while the rest sway or remain still, with spaces of varying sizes between the sperm, each sperm is clearly distinguishable, and the number of motile sperm accounts for 40% of the total.

[0065] Diluent 2: When the semen and diluent are mixed at a 1:1 ratio, microscopic examination reveals that only a few sperm move in a straight line, while the rest sway or remain still. There is almost no space between the sperm, and the number of motile sperm accounts for 20% of the total. When mixed at a 1:4 ratio, all the semen dies.

[0066] 3.4 After freezing and thawing according to the cryopreservation method described in patent application CN114009427A, the viability was observed under a microscope:

[0067] Diluent 1: When the semen and diluent are mixed at a 1:1 ratio, the sperm under a microscope will move in a straight line while the rest will sway or remain still. There is almost no space between the sperm, and the number of motile sperm accounts for 40% of the total. When the diluent is mixed at a 1:4 ratio, the sperm under a microscope will move in a straight line while the rest will sway or remain still. There are spaces of different sizes between the sperm, and each sperm is clearly distinguishable. The number of motile sperm accounts for 30% of the total.

[0068] Diluent 2: When semen and diluent are mixed at a 1:1 ratio, the sperm either sway or remain still, with almost no space between the sperm, and the number of motile sperm accounts for 20% of the total. When mixed at a 1:4 ratio, all the semen dies.

[0069] The above experimental results show that, on the one hand, compared with the known rabbit frozen semen diluent in patent application CN114009427A, the rabbit frozen semen diluent provided by the present invention can effectively reduce the damage to semen caused by low temperature during cryopreservation and improve sperm motility during thawing; on the other hand, compared with the cryopreservation method used in patent application CN114009427A, the rabbit frozen semen diluent provided by the present invention is more conducive to reducing the damage to semen caused by low temperature during cryopreservation and improving sperm motility during thawing under the cryopreservation procedure of Example 2 of the present invention.

[0070] In summary, this invention provides a rabbit frozen semen diluent and a method for cryopreserving rabbit semen. The rabbit frozen semen diluent and method provided by this invention can be used for cryopreserving rabbit semen at low temperatures, effectively reducing the damage to semen caused by low temperatures during cryopreservation, improving sperm motility and the conception rate of female rabbits upon thawing, and has broad application prospects.

Claims

1. A rabbit frozen semen diluent, characterized in that: It contains the following ingredients: tris(hydroxymethyl)aminomethane, citric acid, glucose, sucrose, antibiotics, dimethyl sulfoxide, egg yolk, water, and pH adjuster; The concentrations of tris(hydroxymethyl)aminomethane were 1.900 g / 50 mL, citric acid was 0.990 g / 50 mL, glucose was 0.525 g / 50 mL, sucrose was 0.428 g / 50 mL, kanamycin was 0.004 g / 50 mL, dimethyl sulfoxide was 5 mL / 50 mL, and egg yolk was 10 mL / 50 mL. The pH value of the rabbit frozen semen diluent is 6.0 to 8.

0.

2. The rabbit frozen semen diluent according to claim 1, characterized in that: The pH value of the rabbit frozen semen dilution is 7.

0.

3. The method for preparing the rabbit frozen semen diluent according to any one of claims 1 to 2, characterized in that: The method includes the following steps: mixing tris(hydroxymethyl)aminomethane, citric acid, glucose, sucrose, antibiotics, dimethyl sulfoxide and egg yolk, adding a pH adjuster to adjust the pH value, adding water to make up the volume, and obtaining a diluted solution of rabbit frozen semen.

4. A method for cryopreserving rabbit semen, characterized in that, It includes the following steps: Add the rabbit frozen semen diluent according to any one of claims 1 to 2 to rabbit semen to obtain a mixture, dispense, equilibrate, and freeze.

5. The method according to claim 4, characterized in that, The volume ratio of rabbit semen to rabbit frozen semen diluent is 1:(1-4), the equilibration temperature is 2-6℃, and the time is 0.5-3h; the cryopreservation procedure is as follows: place the equilibrated aliquoted mixture at -85 to -105℃, and then cool it down to -140 to -160℃ at a rate of -1 to -5℃ / min, and store it in a liquid nitrogen tank.

6. The method according to claim 5, characterized in that, The equilibration temperature is 4℃ and the time is 2h; the cryopreservation procedure is as follows: place the equilibrated aliquoted mixture at -95℃, then cool it down to -150℃ at a rate of -3℃ / min, and store it in a liquid nitrogen tank.